PROJECT SUMMARY/ABSTRACT Pediatric brain tumors have recently surpassed leukemia as the most common cause of cancer-related death in children. Immunotherapies leveraging the specificity of cytotoxic T cells have demonstrated unprecedented treatment responses for some malignancies; however, a lack of known targets prevents its application to the treatment of pediatric brain tumors. Medulloblastoma (MB) is the most common malignant pediatric brain tumor and is now understood to include at least four distinct molecular variants. Group 3 MB is the deadliest and among the most prevalent subtypes. Despite the use of aggressive treatments, the overall survival of patients with Group 3 MB remains below 50%. Serious long-term side effects and high relapse rates indicate that more targeted and effective therapies are desperately needed. Thus, the long-term goal of this proposal is to develop an immunologic strategy for treating high-risk MB. In melanoma, the most commonly appreciated immunologic targets are mutated proteins that generate immunogenic epitopes unique to tumor cells (neoantigens). In contrast to melanoma, the prevalence of mutations in pediatric brain tumors is paltry. Epigenetic modifiers predominate the few recurrently mutated genes. In MB, aberrant epigenetic gene regulation is shown to drive transcription patterns reminiscent of pluripotent stem cells and developing neural precursors. Pluripotent stem cells and neural stem cells therefore represent potential cellular sources of developmentally-regulated antigens (Dev Ags) that can be used to prime immune responses. The central hypothesis of this proposal portends that immunologic targeting of aberrantly expressed developmental proteins in Group 3 MB will provide a novel therapeutic platform with enhanced curative potential for these patients. This hypothesis will be tested through three specific aims: 1) Evaluate the capacity of induced pluripotent stem cells (iPSCs) and their spatiotemporally distinct neural progeny to serve as sources of antigen for immunologic targeting of Group 3 MB; 2) Determine the effect of chemotherapy, radiation, and epigenetic modifiers on the expression and anti-tumor efficacy of Dev Ags in Group 3 MB tumors; and 3) Demonstrate the ability of human Dev Ag specific T cells to target human MB tumors in vitro and in a patient-derived xenograft model of Group 3 MB. Aim 1, will utilize an in vitro model of neurodevelopment to analyze the transcriptional overlap of iPSCs and distinct iPSC-derived neural stem and progenitors (iNSPCs) with Group 3 MB and will evaluate the specificity and efficacy for targeting MB using Dev Ag T cells. Aim 2 will evaluate changes in gene expression, T cell specificity, and anti-tumor efficacy following exposure of MB tumors to radiation, chemotherapy, and epigenetic modifiers. Finally, using autologous iPSCs, iNSPCs, immune cells, and MB tumors, the efficacy of Dev Ag T cells in patient-derived xenografts of Group 3 MB will be evaluated. This research is significant in that it will provide an innovative strategy for deriving antigens with the capacity to immunologically target MB and demonstrate the effectiveness of these antigens at directing cytotoxic T cell responses against mouse and human tumors.